Mutant 1,3-propanediol dehydrogenase

ABSTRACT

The present invention relates to mutant 1,3-propanediol dehydrogenase and a novel microorganism that is capable of growing in concentrations of at least 105 g/l 1,3-propanediol, levels normally toxic to wild-type microorganisms. The present invention also provides expression vectors and host cells comprising the mutant 1,3-propanediol dehydrogenase as well as methods for producing 1,3-propanediol comprising the use of cells comprising the mutant 1,3-propanediol dehydrogenase.

FIELD OF THE INVENTION

[0001] The present invention relates to mutant 1,3-propanedioldehydrogenase having an altered Km for 1,3-propanediol. The presentinvention provides the nucleic acid and amino acid sequence of themutant form of 1,3-propanediol dehydrogenase. The present invention alsoprovides expression vectors and host cells comprising mutant1,3-propanediol dehydrogenase.

BACKGROUND OF THE INVENTION

[0002] 1,3-Propanediol is a monomer having potential utility in theproduction of polyester fibers and the manufacture of polyurethanes andcyclic compounds. The production of 1,3-propanediol has been disclosedin U.S. Pat. No. 5,686,276 issued Nov. 11, 1997 and WO 98/21341. Onerepresentative pathway for the production of 1,3-propanediol fromglucose can be accomplished by the following series of steps. Glucose isconverted in a series of steps by enzymes of the glycolytic pathway todihydroxyacetone phosphate (DHAP) and 3-phosphoglyceraldehyde (3-PG).Glycerol is then formed by either hydrolysis of DHAP to dihydroxyacetone(DHA) followed by reduction, or reduction of DHAP to glycerol3-phosphate (G3P) followed by hydrolysis. The hydrolysis step can becatalyzed by any number of cellular phosphatases which are known to bespecific or non-specific with respect to their substrates or theactivity can be introduced into the host by recombination. The reductionstep can be catalyzed by a NAD⁺ (or NADP⁺) linked host enzyme or theactivity can be introduced into the host by recombination. It is notablethat the dha regulon contains a glycerol dehydrogenase (E.C. 1.1.1.6)which catalyzes the reversible reaction of Equation 3.

Glycerol® 3-HP+H₂O  (Equation 1)

3-HP+NADH+H⁺® 1,3-Propanediol+NAD⁺  (Equation 2)

Glycerol+NAD⁺® DHA+NADH+H⁺  (Equation 3)

[0003] Glycerol is converted to 1,3-propanediol via the intermediate3-hydroxypropionaldehye (3-HP) as has been described in detail above.The intermediate 3-HP is produced from glycerol (Equation 1) by adehydratase enzyme which can be encoded by the host or can introducedinto the host by recombination. This dehydratase can be glyceroldehydratase (E.C. 4.2.1.30), diol dehydratase (E.C. 4.2.1.28), or anyother enzyme able to catalyze this transformation. Glycerol dehydrataseis encoded by the dha regulon. 1,3-Propanediol is produced from 3-HP(Equation 2) by a NAD⁺-(or NADP⁺) linked host enzyme or the activity canintroduced into the host by recombination. This final reaction in theproduction of 1,3-propanediol can be catalyzed by 1,3-propanedioldehydrogenase (E.C. 1.1.1.202) or other alcohol dehydrogenases.

[0004] In Klebsiella pneumoniae and Citrobacter freundii, the genesencoding the functionally linked activities of glycerol dehydratase(dhaB), 1,3-propanediol oxidoreductase (dhaT), glycerol dehydrogenase(dhaD), and dihydroxyacetone kinase (dhaK) are encompassed by the dharegulon. The dha regulons from Citrobacter and Klebsiella have beenexpressed in Escherichia coli and have been shown to convert glycerol to1,3-propanediol.

[0005] Nucleic acid and amino acid sequences for a 1,3-propanedioldehydrogenase that have been disclosed in the art, including Klebsiellapneumoniae GenBank accession # U30903 (Williard, 1994, “Investigation ofthe Klebsiella pneumoniae 1,3-propanediol pathway: Characterization andexpression of glycerol dehydratase and 1,3-propanediol oxidoreductase”Thesis Chemical Engineering, University of Wisconsin-Madison);Citrobacter freundii GenBank accession # U09771 (Daniel, R. et al.,1995, Purification of 1,3-propanediol dehydrogenase from Citrobacterfreundii: cloning, sequencing, and overexpression of the correspondinggene in Escherichia coli. J. Bacteriol. 177:2151-2156); and Clostridiumpasteurianum GenBank accession # AF006034 (Luers,F. et al., 1997,Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum:cloning and expression of the gene encoding 1,3-propanedioldehydrogenase. FEMS Microbiol. Lett. 154:337-345).

SUMMARY OF THE INVENTION

[0006] The present invention relates to the discovery of a mutant formof 1,3-propanediol dehydrogenase (PDD) isolated from a derivative ofE.blattae capable of growth in the presence of at least 105 g/l1,3-propanediol, levels normally toxic to wild-type E.blattae. Thepresent invention is therefore based in part upon the discovery that themutant form of PDD is associated with E.blattae's resistance to normallytoxic levels of 1,3-propanediol. The present invention is also based inpart upon the finding that this mutant PDD has an altered Km for1,3-propanediol and NAD.

[0007] Accordingly, the present invention provides a mutant PDD having aKm for 1,3-propanediol that is increased over the wild-type PDD Km for1,3-propanediol. In one embodiment, the Km of the mutant PDD is about 3times the Km of wild-type PDD for 1,3-propanediol. In anotherembodiment, the mutant PDD has a Km of about 80 mM for 1,3-propanediol.In a further embodiment, the mutant PDD is obtainable from E.blattaeATCC accession number PTA-92.

[0008] In yet another embodiment of the present invention, the mutantPDD comprises a mutation corresponding to residue His105 to Leu inE.blatte PDD as shown in FIG. 3. In an additional embodiment, the mutantPDD comprises the amino acid shown in SEQ ID NO:2 and is encoded bynucleic acid having the sequence as shown in SEQ ID NO:1.

[0009] The present invention also provides expression vectors and hostcells comprising the isolated nucleic acid having the sequence as shownin SEQ ID NO:1. In one embodiment, the host cell includes Citrobacter,Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus,Aspergillus, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces,Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor,Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus,Streptomyces and Pseudomonas.

[0010] In an additional aspect, the present invention relates to methodsfor producing 1,3-propanediol comprising the use of a microorganismcomprising mutant PDD.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 provides the nucleic acid sequence (SEQ ID NO:1) for themutant 1,3-propanediol dehydrogenase (PDD).

[0012]FIG. 2 provides the amino acid sequence (SEQ ID NO:2) for themutant 1,3-propanediol dehydrogenase (PDD).

[0013]FIG. 3 provides an amino acid alignment of PDDs from variousmicroorganisms. Eb_GEBT represents the ATCC deposited E.blattae mutantPDD, Eb_(—)429T and Eb_(—)907T are wild-type E.blattae (ATCC accessionnumber 33429); Kpn is Klebsiella pneumoniae (GenBank accession #U30903); Cfu is Citrobacter freundii (GenBank accession number U09771)and Cpast is Clostridium pasteurianum (GenBank accession numberAF006034).

DESCRIPTION OF THE MICROORGANISM DEPOSITS MADE UNDER THE BUDAPEST TREATY

[0014] Applicants have made the following biological deposits under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for the Purposes of Patent Procedure:Depositor Identification International Depository Reference DesignationDate of Deposit Escherichia blattae PTA-92 May 19, 1999 33429 derivative

DETAILED DESCRIPTION Definitions

[0015] The terms “1,3-propanediol dehydrogenase” or “PDD” (also known inthe art as “oxidoreductase”) refer to the polypeptide(s) responsible foran enzyme activity that is capable of catalyzing the reduction of3-hydroxypropionaldehyde to 1,3-propanediol. 1,3-Propanedioldehydrogenase includes, for example, the polypeptide encoded by the dhaTgene. The present invention encompasses 3-propanediol dehydrogenase fromany source including, but not limited to E.blatte, K.pneumoniae,C.freundii, C.pasteurianum.

[0016] As used herein, the term “mutant” or “mutation” refers to anygenetic change that occurs in the nucleic acid of a microorganism andmay or may not reflect a phenotypic change within the microorganism. Amutation may comprise a single base pair change, deletion or insertion;a mutation may comprise a change, deletion or insertion in a largenumber of base pairs; a mutation may also comprise a change in a largeregion of DNA, such as through duplication or inversion. The amino acidsequence of a mutant 1,3-propanediol dehydrogenase can be derived from aprecursor 1,3-propanediol dehydrogenase by the substitution, deletion orinsertion of one or more amino acids of the naturally occurring1,3-propanediol dehydrogenase. Methods for modifying genes (e.g.,through site-directed oligonucleotide mutagenesis) have been describedin the art.

[0017] The phrase “corresponding to” as used herein refers to the aminoacid relatedness among 1,3-propanediol dehydrogenases as exemplified byFIG. 3. Specific residues discussed herein refer to an amino acidresidue number which references the number assigned to the E.blatte GEBPDD shown in FIG. 3. The mutation of His to Leu is shown at residue 105in FIG. 3. FIG. 3 illustrates that 1,3-propanediol dehydrogenases from avariety of microbial sources can be aligned using the algorithmCLUSTALW. The invention is not limited to the mutation of the E.blattaePDD shown in FIGS. 1 and 2, or the E.blattae deposited with the ATCC andhaving accession number PTA-92 but encompasses all PDDs containing aminoacid residues at positions which are equivalent to the particularidentified residue in E.blattae. A residue is equivalent if it is eitherhomologous (i.e., corresponds in position for either the primary ortertiary structure) or analogous to a specific residue or portion ofthat residue in E.blattae PDD (i.e., having the same or similarfunctional capacity to combine, react, or interact chemically orstructurally).

[0018] In order to establish homology to primary structure, the aminoacid sequence of a PDD is directly compared to the E.blattae PDD primarysequence (shown in FIG. 2) and particularly to a set of residues knownto be invariant to all PDDs for which sequences are known (see, e.g.,FIG. 3). The present invention encompasses the equivalent residue changein all sources of 1,3-propanediol dehydrogenase as long as the mutantform is able to alter the Km of the activity for 1,3-propanediol. In apreferred embodiment, the Km of the mutant form is increased for1,3-propanediol. The nucleic acid sequence of SEQ ID NO:1 was obtainedvia PCR techniques. Such techniques are often characterized byinadvertent PCR generated sequence error. Therefore, the presentinvention also encompasses the 1,3-propanediol dehydrogenase ofE.blattae having ATCC accession number PTA-92 and correspondingmutations in other microbial sources of the 1,3-propanedioldehydrogenases.

[0019] The term “Km” refers to affinity of the enzyme for the substrate.A high Km reflects a low affinity; a low Km reflects a high affinity.

[0020] The terms “carbon substrate” and “carbon source” refer to acarbon source capable of being metabolized by host organisms of thepresent invention and particularly carbon sources selected from thegroup consisting of monosaccharides, oligosaccharides, polysaccharides,and one-carbon substrates or mixtures thereof.

[0021] The terms “host cell” or “host organism” refer to a microorganismcapable of receiving foreign or heterologous genes and of expressingthose genes to produce an active gene product.

[0022] As used herein, “nucleic acid” refers to a nucleotide orpolynucleotide sequence, and fragments or portions thereof, and to DNAor RNA of genomic or synthetic origin which may be double-stranded orsingle-stranded, whether representing the sense or antisense strand. Theterms “native” and “wild-type” refer to a gene as found in nature withits own regulatory sequences. As used herein “amino acid” refers topeptide or protein sequences or portions thereof.

[0023] The term “expression” refers to the transcription and translationto gene product from a gene coding for the sequence of the gene product.

[0024] The terms “plasmid”, “vector”, and “cassefte” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

[0025] The terms “isolated” or “purified” as used herein refer to anucleic acid or amino acid that is removed from at least one componentwith which it is naturally associated.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention relates to mutant 1,3-propanedioldehydrogenase (PDD) characterized by having an increased Km for 1,3propanediol.

[0027] I. PDD Sequences

[0028] Polynucleotide sequence as shown in SEQ ID NO:1 encodes the1,3-propanediol dehydrogenase (SEQ ID NO:2) having the mutation of Histo Leu at residue 105 as shown in FIG. 3. As will be understood by theskilled artisan, due to the degeneracy of the genetic code, a variety ofpolynucleotides can encode SEQ ID NO:2. The present inventionencompasses all such polynucleotides. The present invention encompassesnucleic acid encoding PDD comprising a mutation corresponding toE.blatte residue His 105 to Leu as shown in FIG. 3. The nucleic acid andamino acid sequence for PDD from K.pneumoniae is given in GenBankaccession number U30903; PDD from C. freundii is given in GenBankaccession number U09771; for PDD from C.pasteurianum is given in GenBankaccession number AF00034. The present invention also encompasses mutantPDD obtainable from E.blattae having ATCC accession number PTA-92.

[0029] Methods of obtaining desired genes from a microbial genome arecommon and well known in the art of molecular biology. For example, ifthe sequence of the gene is known, suitable genomic libraries may becreated by restriction endonuclease digestion and may be screened withprobes complementary to the desired gene sequence. Once the sequence isisolated, the DNA may be amplified using standard primer directedamplification methods such as polymerase chain reaction (PCR) (U.S. Pat.No. 4,683,202) to obtain amounts of DNA suitable for transformationusing appropriate vectors.

[0030] Alternatively, methods of using cosmid vectors for thetransformation of suitable bacterial hosts are well described inSambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition(1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbon, N.Y.(1989).

[0031] Methods of making mutations in PDD genes are known to the skilledartisan and include for example site-directed mutagenesis, proceduresdescribed in United States patent U.S. Pat. No. 4,760,025 issued Jul.26, 1988.

[0032] Vectors and Expression Cassettes

[0033] The present invention provides a variety of vectors andtransformation and expression cassettes suitable for the cloning,transformation and expression of mutant PDD as well as other proteinsassociated with 1,3-propanediol production into a suitable host cell.Suitable vectors will be those which are compatible with the bacteriumemployed. Suitable vectors can be derived, for example, from a bacteria,a virus (such as bacteriophage T7 or a M-13 derived phage), a cosmid, ayeast or a plant. Protocols for obtaining and using such vectors areknown to those in the art. (Sambrook et al., Molecular Cloning: ALaboratory Manual—volumes 1,2,3 (Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., (1989)).

[0034] Typically, the vector or cassette contains sequences directingtranscription and translation of the relevant gene, a selectable marker,and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene whichharbors transcriptional initiation controls and a region 3′ of the DNAfragment which controls transcriptional termination. It is mostpreferred when both control regions are derived from genes homologous tothe transformed host cell although it is to be understood that suchcontrol regions need not be derived from the genes native to thespecific species chosen as a production host.

[0035] Initiation control regions or promoters, which are useful todrive expression of PDD in the desired host cell, are numerous andfamiliar to those skilled in the art. Virtually any promoter capable ofdriving these genes is suitable for the present invention including butnot limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1,TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces);AOX1 (useful for expression in Pichia); and lac, trp, IP_(L), IP_(R),T7, tac, and trc (useful for expression in E. coli).

[0036] Termination control regions may also be derived from variousgenes native to the preferred hosts. Optionally, a termination site maybe unnecessary, however, it is most preferred if included.

[0037] For effective expression of the instant enzymes, DNA encoding theenzymes are linked operably through initiation codons to selectedexpression control regions such that expression results in the formationof the appropriate messenger RNA.

[0038] Transformation of Suitable Hosts and Expression of PDD

[0039] Once suitable cassettes are constructed they are used totransform appropriate host cells. Introduction of the cassettecontaining mutant 1,3-propanediol dehydrogenase, either separately ortogether with other proteins necessary for the production of1,3-propanediol, into the host cell may be accomplished by knownprocedures such as by transformation (e.g., using calcium-permeabilizedcells, electroporation) or by transfection using a recombinant phagevirus. (Sambrook et al., supra.).

[0040] Host Cells

[0041] Suitable host cells for the recombinant production of1,3-propanediol may be either prokaryotic or eukaryotic and will belimited only by the host cell ability to express active enzymes.Preferred hosts will be those typically useful for production ofglycerol or 1,3-propanediol such as Citrobacter, Enterobacter,Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus,Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Pichia,Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis,Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces andPseudomonas. Most preferred in the present invention are E. coli,Klebsiella species and Saccharomyces species.

[0042] Media and Carbon Substrates

[0043] Fermentation media in the present invention must contain suitablecarbon substrates. Suitable substrates may include but are not limitedto monosaccharides such as glucose and fructose, oligosaccharides suchas lactose or sucrose, polysaccharides such as starch or cellulose, ormixtures thereof, and unpurified mixtures from renewable feedstocks suchas cheese whey permeate, cornsteep liquor, sugar beet molasses, andbarley malt. Additionally, the carbon substrate may also be one-carbonsubstrates such as carbon dioxide, or methanol for which metabolicconversion into key biochemical intermediates has been demonstrated.

[0044] Preferred carbon substrates are monosaccharides,oligosaccharides, polysaccharides, and one-carbon substrates. Morepreferred are sugars such as glucose, fructose, sucrose and singlecarbon substrates such as methanol and carbon dioxide. Most preferred isglucose.

[0045] In addition to an appropriate carbon source, fermentation mediamust contain suitable minerals, salts, cofactors, buffers and othercomponents, known to those skilled in the art, suitable for the growthof the cultures and promotion of the enzymatic pathway necessary forglycerol production. Particular attention is given to Co(II) saltsand/or vitamin B₁₂ or precursors thereof.

[0046] Culture Conditions

[0047] Typically, cells are grown at 30° C. in appropriate media.Preferred growth media in the present invention are common commerciallyprepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD)broth or Yeast Malt Extract (YM) broth. Other defined or syntheticgrowth media may also be used and the appropriate medium for growth ofthe particular microorganism will be known by someone skilled in the artof microbiology or fermentation science. The use of agents known tomodulate catabolite repression directly or indirectly, e.g., cyclicadenosine 2′:3′-monophosphate or cyclic adenosine 2′:5′-monophosphate,may also be incorporated into the reaction media. Similarly, the use ofagents known to modulate enzymatic activities (e.g., sulphites,bisulphites and alkalis) that lead to enhancement of glycerol productionmay be used in conjunction with or as an alternative to geneticmanipulations.

[0048] Suitable pH ranges for the fermentation are between pH 5.0 to pH9.0, where pH 6.0 to pH 8.0 is preferred as range for the initialcondition.

[0049] Reactions may be performed under aerobic or anaerobic conditionswhere anaerobic or microaerobic conditions are preferred.

[0050] The manner and method of carrying out the present invention maybe more fully understood by those of skill in the art by reference tothe following examples, which examples are not intended in any manner tolimit the scope of the present invention or of the claims directedthereto.

EXAMPLES

[0051] Example 1 describes the kinetic changes associated with themutant PDD shown in SEQ ID NO:2.

[0052] Materials and Methods

[0053] Strains—Wild type ATCC 33429, E.blattae comprising the mutant PDDATCC accession number PTA-92.

[0054] Growth—Cells were grown in a complex medium at 30C 500 ml in a2800 ml fernbach with shaking at 225 rpm for 20 hr. The medium consistsof KH2PO4, 5.4 g/L; (NH4)2SO4, 1.2 g/L; MgSO47H2O, 0.4 g/L; yeastextract, 2.0 g/L; tryptone, 2.0 g/L; and glycerol, 9.2 g/L in tap water.The pH was adjusted to 7.1 with KOH before autoclaving (Honda, et al.,1980, J. Bacteriol, 143:1458-1465).

[0055] Extract prep—Cells were harvested by centrifugation with care toavoid anaerobic conditions. Pellets were resuspended in 100 mM TricinepH 8.2 containing 50 mM KCl and 1 mM DTT. Cells were disrupted bypassage through a French pressure cell. Crude extracts were clarified bycentrifugation at 20K×g for 20 min followed by 100K×g for 1 hr to yieldthe high speed supernatant (HSS) fraction.

[0056] Assays—the assay for PDD was performed as described by Johnson,E. A. et al., 1987, J. Bacteriol. 169:2050-2054.

[0057] Partial purification of PDD—HSS was separated on a 16×100 Poros20HQ column. The buffers were A, 50 mM HEPES, pH 7.4 containing 100 uMMnCl and B, A buffer containing 500 mM KCl. The column was loaded anddeveloped at 10 ml/min. The gradient was 10 CV wash, a linear gradientto 70% B in 10 CV, and 1 CV to 100% B. The activity was detected in thevery early fractions of the gradient. Pooled column fractions of the33429 strain were used as collected for assays after the addition ofadditional of DTT to 1 mM. The active fractions from strain GEB031 werepooled and concentrated on a PM30 membrane and used as concentratedafter the addition of additional 1 mM DTT. Strain GD (U/mg) PDD (U/mg)Ratio GD/PDD 33429 0.64 0.22 2.9 GEB031 0.79 0.08 9.9

[0058] PDD Kinetics—The results are shown below. Strain Km (mMPropanediol) Km (uM NAD) 33429 28 57

[0059] Example 2: Cloning and sequencing the 1,3-propanedioldehydrogenase genes (dhaT) from E. blattae.

[0060] The dhaT genes were amplified by PCR from genomic DNA from E.blattae as template DNA using synthetic primers (primer 1 and primer 2)based on the K. pneumoniae dhaT sequence and incorporating an XbaI siteat the 5′ end and a BamHI site at the 3′ end. The product was subclonedinto pCR-Blunt II-TOPO (Invitrogen). The cloning dhaT were thensequenced was standard techniques.

[0061] The results of the DNA sequencing are given in SEQ ID NO:1 andSEQ ID NO:2. Primer 1 5′ TCTGATACGGGATCCTCAGAATGCCTGGCGGAAAAT3′ Primer 25′ GCGCCGTCTAGAATTATGAGCTATCGTATGTTTGATTATCTG3′

[0062] As will be readily understood by the skilled artisan, nucleicacid sequence generated via PCR methods may comprise inadvertent errors.The present invention also encompasses nucleic acid encoding PDDobtainable from E.blattae having ATCC accession number PTA-92.

[0063] All references cited herein, including patents, patentapplications, sequences and publications are hereby incorporated intheir entirety by reference.

1 12 1 1164 DNA Escherichia blattae 1 atgagctatc gtatgtttga ttatctggttccaaatgtra acttctttgg cccgggcgcc 60 gtttctgttg ttggccagcg ctgccagctgctggggggta aaaaagccct gctggtgacc 120 gataagggcc tgcgcgccat taaagacggtgctgtcgatc agaccgtgaa gcacctgaaa 180 gccgccggta ttgaggtggt cattttcgacggggtcgagc cgaacccgaa agacaccaac 240 gtgctcgacg gcctggccat gttccgtaaagagcagtgcg acatgataat caccgtcggc 300 ggcggcagcc cgctcgactg cggtaaaggcattggtattg cggccaccca cccgggtgat 360 ctgtacagct atgccggtat cgaaacactcaccaacccgc tgccgcccat tattgcggtc 420 aacaccaccg ccgggaccgc cagcgaagtcacccgccact gcgtgctgac taacaccaaa 480 accaaagtaa aatttgtgat tgtcagctggcgcaacctgc cttccgtctc cattaacgat 540 ccgctgctga tgatcggcaa gcccgccgggctgaccgccg ccaccggtat ggatgccctg 600 acccacgcgg tagaggccta tatctccaaagacgccaacc cggttaccga tgcctctgct 660 attcaggcca tcaaactgat tgccaccaacttgcgccagg ccgtcgccct ggggaccaac 720 ctcaaagccc gtgaaaacat ggcctgcgcctctctgctgg ccgggatggc ctttaacaac 780 gccaacctgg gctatgttca cgccatggctcaccagctgg gcggcctgta cgacatggcc 840 cacggggtgg cgaacgcggt cctgctgccccatgtctgcc gctataacct gattgccaac 900 ccggaaaaat ttgccgatat cgccacctttatgggggaaa acaccaccgg tctttccacc 960 atggacgcag cggagctggc catcagcgccattgcccgtc tgtctaaaga tgtcgggatc 1020 ccgcagcacc tgcgtgaact gggggtaaaagaggccgact tcccgtacat ggcagaaatg 1080 gccctgaaag acggcaacgc cttctctaacccgcgcaaag ggaacgaaaa agagattgcc 1140 gacattttcc gccaggcatt ctga 1164 2387 PRT Escherichia blattae 2 Met Ser Tyr Arg Met Phe Asp Tyr Leu ValPro Asn Val Asn Phe Phe 1 5 10 15 Gly Pro Gly Ala Val Ser Val Val GlyGln Arg Cys Gln Leu Leu Gly 20 25 30 Gly Lys Lys Ala Leu Leu Val Thr AspLys Gly Leu Arg Ala Ile Lys 35 40 45 Asp Gly Ala Val Asp Gln Thr Val LysHis Leu Lys Ala Ala Gly Ile 50 55 60 Glu Val Val Ile Phe Asp Gly Val GluPro Asn Pro Lys Asp Thr Asn 65 70 75 80 Val Leu Asp Gly Leu Ala Met PheArg Lys Glu Gln Cys Asp Met Ile 85 90 95 Ile Thr Val Gly Gly Gly Ser ProLeu Asp Cys Gly Lys Gly Ile Gly 100 105 110 Ile Ala Ala Thr His Pro GlyAsp Leu Tyr Ser Tyr Ala Gly Ile Glu 115 120 125 Thr Leu Thr Asn Pro LeuPro Pro Ile Ile Ala Val Asn Thr Thr Ala 130 135 140 Gly Thr Ala Ser GluVal Thr Arg His Cys Val Leu Thr Asn Thr Lys 145 150 155 160 Thr Lys ValLys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 165 170 175 Ser IleAsn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Gly Leu Thr 180 185 190 AlaAla Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile 195 200 205Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala Ile Gln Ala Ile 210 215220 Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val Ala Leu Gly Thr Asn 225230 235 240 Leu Lys Ala Arg Glu Asn Met Ala Cys Ala Ser Leu Leu Ala GlyMet 245 250 255 Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val His Ala Met AlaHis Gln 260 265 270 Leu Gly Gly Leu Tyr Asp Met Ala His Gly Val Ala AsnAla Val Leu 275 280 285 Leu Pro His Val Cys Arg Tyr Asn Leu Ile Ala AsnPro Glu Lys Phe 290 295 300 Ala Asp Ile Ala Thr Phe Met Gly Glu Asn ThrThr Gly Leu Ser Thr 305 310 315 320 Met Asp Ala Ala Glu Leu Ala Ile SerAla Ile Ala Arg Leu Ser Lys 325 330 335 Asp Val Gly Ile Pro Gln His LeuArg Glu Leu Gly Val Lys Glu Ala 340 345 350 Asp Phe Pro Tyr Met Ala GluMet Ala Leu Lys Asp Gly Asn Ala Phe 355 360 365 Ser Asn Pro Arg Lys GlyAsn Glu Lys Glu Ile Ala Asp Ile Phe Arg 370 375 380 Gln Ala Phe 385 3 36DNA Artificial Sequence primer 3 tctgatacgg gatcctcaga atgcctggcg gaaaat36 4 42 DNA Artificial Sequence primer 4 gcgccgtcta gaattatgagctatcgtatg tttgattatc tg 42 5 1164 DNA Escherichia blattae 5 atgagctatcgtatgtttga ttatctggtt ccaaatgtga acttctttgg cccgggcgcc 60 gtttctgttgttggccagcg ctgccagctg ctggggggta aaaaagccct gctggtgacc 120 gataagggcctgcgcgccat taaagacggt gctgtcgatc agaccgtgaa gcacctgaaa 180 gccgccggtattgaggtggt cattttcgac ggggtcgagc cgaacccgaa agacaccaac 240 gtgctcgacggcctggccat gttccgtaaa gagcagtgcg acatgataat caccgtcggc 300 ggcggcagcccgcacgactg cggtaaaggc attggtattg cggccaccca cccgggtgat 360 ctgtacagctatgccggtat cgaaacactc accaacccgc tgccgcccat tattgcggtc 420 aacaccaccgccgggaccgc cagcgaagtc acccgccact gcgtgctgac taacaccaaa 480 accaaagtaaaatttgtgat tgtcagctgg cgcaacctgc cttccgtctc cattaacgat 540 ccgctgctgatgatcggcaa gcccgccggg ctgaccgccg ccaccggtat ggatgccctg 600 acccacgcggtagaggccta tatctccaaa gacgccaacc cggttaccga tgcctctgct 660 attcaggccatcaaactgat tgccaccaac ttgcgccagg ccgtcgccct ggggaccaac 720 ctcaaagcccgtgaaaacat ggcctgcgcc tctctgctgg ccgggatggc ctttaacaac 780 gccaacctgggctatgttca cgccatggct caccagctgg gcggcctgta cgacatggcc 840 cacggggtggcgaacgcggt cctgctgccc catgtctgcc gctataacct gattgccaac 900 ccggaaaaatttgccgatat cgccaccttt atgggggaaa acaccaccgg tctttccacc 960 atggacgcagcggagctggc catcagcgcc attgcccgtc tgtctaaaga tgtcgggatc 1020 ccgcagcacctgcgtgaact gggggtaaaa gaggccgact tcccgtacat ggcagaaatg 1080 gccctgaaagacggcaacgc cttctctaac ccgcgcaaag ggaacgaaaa agagattgcc 1140 gacattttccgccaggcatt ctga 1164 6 1164 DNA Escherichia blattae 6 atgagctatcgtatgtttga ttatctggtt ccaaatgtra acttctttgg cccgggcgcc 60 gtttctgttgttggccagcg ctgccagctg ctggggggta aaaaagccct gctggtgacc 120 gataagggcctgcgcgccat taaagacggt gctgtcgatc agaccgtgaa gcacctgaaa 180 gccgccggtattgaggtggt cattttcgac ggggtcgagc cgaacccgaa agacaccaac 240 gtgctcgacggcctggccat gttccgtaaa gagcagtgcg acatgataat caccgtcggc 300 ggcggcagcccgctcgactg cggtaaaggc attggtattg cggccaccca cccgggtgat 360 ctgtacagctatgccggtat cgaaacactc accaacccgc tgccgcccat tattgcggtc 420 aacaccaccgccgggaccgc cagcgaagtc acccgccact gcgtgctgac taacaccaaa 480 accaaagtaaaatttgtgat tgtcagctgg cgcaacctgc cttccgtctc cattaacgat 540 ccgctgctgatgatcggcaa gcccgccggg ctgaccgccg ccaccggtat ggatgccctg 600 acccacgcggtagaggccta tatctccaaa gacgccaacc cggttaccga tgcctctgct 660 attcaggccatcaaactgat tgccaccaac ttgcgccagg ccgtcgccct ggggaccaac 720 ctcaaagcccgtgaaaacat ggcctgcgcc tctctgctgg ccgggatggc ctttaacaac 780 gccaacctgggctatgttca cgccatggct caccagctgg gcggcctgta cgacatggcc 840 cacggggtggcgaacgcggt cctgctgccc catgtctgcc gctataacct gattgccaac 900 ccggaaaaatttgccgatat cgccaccttt atgggggaaa acaccaccgg tctttccacc 960 atggacgcagcggagctggc catcagcgcc attgcccgtc tgtctaaaga tgtcgggatc 1020 ccgcagcacctgcgtgaact gggggtaaaa gaggccgact tcccgtacat ggcagaaatg 1080 gccctgaaagacggcaacgc cttctctaac ccgcgcaaag ggaacgaaaa agagattgcc 1140 gacattttccgccaggcatt ctga 1164 7 387 PRT Escherichia blattae 7 Met Ser Tyr Arg MetPhe Asp Tyr Leu Val Pro Asn Val Asn Phe Phe 1 5 10 15 Gly Pro Gly AlaVal Ser Val Val Gly Gln Arg Cys Gln Leu Leu Gly 20 25 30 Gly Lys Lys AlaLeu Leu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 35 40 45 Asp Gly Ala ValAsp Gln Thr Val Lys His Leu Lys Ala Ala Gly Ile 50 55 60 Glu Val Val IlePhe Asp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 65 70 75 80 Val Leu AspGly Leu Ala Met Phe Arg Lys Glu Gln Cys Asp Met Ile 85 90 95 Ile Thr ValGly Gly Gly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 100 105 110 Ile AlaAla Thr His Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 115 120 125 ThrLeu Thr Asn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala 130 135 140Gly Thr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 145 150155 160 Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val165 170 175 Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Gly LeuThr 180 185 190 Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu AlaTyr Ile 195 200 205 Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala IleGln Ala Ile 210 215 220 Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val AlaLeu Gly Thr Asn 225 230 235 240 Leu Lys Ala Arg Glu Asn Met Ala Cys AlaSer Leu Leu Ala Gly Met 245 250 255 Ala Phe Asn Asn Ala Asn Leu Gly TyrVal His Ala Met Ala His Gln 260 265 270 Leu Gly Gly Leu Tyr Asp Met AlaHis Gly Val Ala Asn Ala Val Leu 275 280 285 Leu Pro His Val Cys Arg TyrAsn Leu Ile Ala Asn Pro Glu Lys Phe 290 295 300 Ala Asp Ile Ala Thr PheMet Gly Glu Asn Thr Thr Gly Leu Ser Thr 305 310 315 320 Met Asp Ala AlaGlu Leu Ala Ile Ser Ala Ile Ala Arg Leu Ser Lys 325 330 335 Asp Val GlyIle Pro Gln His Leu Arg Glu Leu Gly Val Lys Glu Ala 340 345 350 Asp PhePro Tyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 355 360 365 SerAsn Pro Arg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg 370 375 380Gln Ala Phe 385 8 387 PRT Escherichia blattae 8 Met Ser Tyr Arg Met PheAsp Tyr Leu Val Pro Asn Val Asn Phe Phe 1 5 10 15 Gly Pro Gly Ala ValSer Val Val Gly Gln Arg Cys Gln Leu Leu Gly 20 25 30 Gly Lys Lys Ala LeuLeu Val Thr Asp Lys Gly Leu Arg Ala Ile Lys 35 40 45 Asp Gly Ala Val AspGln Thr Val Lys His Leu Lys Ala Ala Gly Ile 50 55 60 Glu Val Val Ile PheAsp Gly Val Glu Pro Asn Pro Lys Asp Thr Asn 65 70 75 80 Val Leu Asp GlyLeu Ala Met Phe Arg Lys Glu Gln Cys Asp Met Ile 85 90 95 Ile Thr Val GlyGly Gly Ser Pro Leu Asp Cys Gly Lys Gly Ile Gly 100 105 110 Ile Ala AlaThr His Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 115 120 125 Thr LeuThr Asn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala 130 135 140 GlyThr Ala Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 145 150 155160 Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 165170 175 Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Gly Leu Thr180 185 190 Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala TyrIle 195 200 205 Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala Ile GlnAla Ile 210 215 220 Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val Ala LeuGly Thr Asn 225 230 235 240 Leu Lys Ala Arg Glu Asn Met Ala Cys Ala SerLeu Leu Ala Gly Met 245 250 255 Ala Phe Asn Asn Ala Asn Leu Gly Tyr ValHis Ala Met Ala His Gln 260 265 270 Leu Gly Gly Leu Tyr Asp Met Ala HisGly Val Ala Asn Ala Val Leu 275 280 285 Leu Pro His Val Cys Arg Tyr AsnLeu Ile Ala Asn Pro Glu Lys Phe 290 295 300 Ala Asp Ile Ala Thr Phe MetGly Glu Asn Thr Thr Gly Leu Ser Thr 305 310 315 320 Met Asp Ala Ala GluLeu Ala Ile Ser Ala Ile Ala Arg Leu Ser Lys 325 330 335 Asp Val Gly IlePro Gln His Leu Arg Glu Leu Gly Val Lys Glu Ala 340 345 350 Asp Phe ProTyr Met Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 355 360 365 Ser AsnPro Arg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg 370 375 380 GlnAla Phe 385 9 387 PRT Escherichia blattae 9 Met Ser Tyr Arg Met Phe AspTyr Leu Val Pro Asn Val Asn Phe Phe 1 5 10 15 Gly Pro Gly Ala Val SerVal Val Gly Gln Arg Cys Gln Leu Leu Gly 20 25 30 Gly Lys Lys Ala Leu LeuVal Thr Asp Lys Gly Leu Arg Ala Ile Lys 35 40 45 Asp Gly Ala Val Asp GlnThr Val Lys His Leu Lys Ala Ala Gly Ile 50 55 60 Glu Val Val Ile Phe AspGly Val Glu Pro Asn Pro Lys Asp Thr Asn 65 70 75 80 Val Leu Asp Gly LeuAla Met Phe Arg Lys Glu Gln Cys Asp Met Ile 85 90 95 Ile Thr Val Gly GlyGly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 100 105 110 Ile Ala Ala ThrHis Pro Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 115 120 125 Thr Leu ThrAsn Pro Leu Pro Pro Ile Ile Ala Val Asn Thr Thr Ala 130 135 140 Gly ThrAla Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 145 150 155 160Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 165 170175 Ser Ile Asn Asp Pro Leu Leu Met Ile Ser Lys Pro Ala Gly Leu Thr 180185 190 Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile195 200 205 Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ser Ala Ile Gln AlaIle 210 215 220 Lys Leu Ile Ala Thr Asn Leu Arg Gln Ala Val Ala Leu GlyThr Asn 225 230 235 240 Leu Lys Ala Arg Glu Asn Met Ala Cys Ala Ser LeuLeu Ala Gly Met 245 250 255 Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val HisAla Met Ala His Gln 260 265 270 Leu Gly Gly Leu Tyr Asp Met Ala His GlyVal Ala Asn Ala Val Leu 275 280 285 Leu Pro His Val Cys Arg Tyr Asn LeuIle Ala Asn Pro Glu Lys Phe 290 295 300 Ala Asp Ile Ala Thr Phe Met GlyGlu Asn Thr Thr Gly Leu Ser Thr 305 310 315 320 Met Asp Ala Ala Glu LeuAla Ile Ser Ala Ile Ala Arg Leu Ser Lys 325 330 335 Asp Val Gly Ile ProGln His Leu Arg Glu Leu Gly Val Lys Glu Ala 340 345 350 Asp Phe Pro TyrMet Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 355 360 365 Ser Asn ProArg Lys Gly Asn Glu Lys Glu Ile Ala Asp Ile Phe Arg 370 375 380 Gln AlaPhe 385 10 387 PRT Klebsiella pneumoniae 10 Met Ser Tyr Arg Met Phe AspTyr Leu Val Pro Asn Val Asn Phe Phe 1 5 10 15 Gly Pro Asn Ala Ile SerVal Val Gly Glu Arg Cys Gln Leu Leu Gly 20 25 30 Gly Lys Lys Ala Leu LeuVal Thr Asp Lys Gly Leu Arg Ala Ile Lys 35 40 45 Asp Gly Ala Val Asp LysThr Leu His Tyr Leu Arg Glu Ala Gly Ile 50 55 60 Glu Val Ala Ile Phe AspGly Val Glu Pro Asn Pro Lys Asp Thr Asn 65 70 75 80 Val Arg Asp Gly LeuAla Val Phe Arg Arg Glu Gln Cys Asp Ile Ile 85 90 95 Val Thr Val Gly GlyGly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 100 105 110 Ile Ala Ala ThrHis Glu Gly Asp Leu Tyr Gln Tyr Ala Gly Ile Glu 115 120 125 Thr Leu ThrAsn Pro Leu Pro Pro Ile Val Ala Val Asn Thr Thr Ala 130 135 140 Gly ThrAla Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Glu 145 150 155 160Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 165 170175 Ser Ile Asn Asp Pro Leu Leu Met Ile Gly Lys Pro Ala Ala Leu Thr 180185 190 Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile195 200 205 Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ala Ala Met Gln AlaIle 210 215 220 Arg Leu Ile Ala Arg Asn Leu Arg Gln Ala Val Ala Leu GlySer Asn 225 230 235 240 Leu Gln Ala Arg Glu Asn Met Ala Tyr Ala Ser LeuLeu Ala Gly Met 245 250 255 Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val HisAla Met Ala His Gln 260 265 270 Leu Gly Gly Leu Tyr Asp Met Pro His GlyVal Ala Asn Ala Val Leu 275 280 285 Leu Pro His Val Ala Arg Tyr Asn LeuIle Ala Asn Pro Glu Lys Phe 290 295 300 Ala Asp Ile Ala Glu Leu Met GlyGlu Asn Ile Thr Gly Leu Ser Thr 305 310 315 320 Leu Asp Ala Ala Glu LysAla Ile Ala Ala Ile Thr Arg Leu Ser Met 325 330 335 Asp Ile Gly Ile ProGln His Leu Arg Asp Leu Gly Val Lys Glu Ala 340 345 350 Asp Phe Pro TyrMet Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 355 360 365 Ser Asn ProArg Lys Gly Asn Glu Gln Glu Ile Ala Ala Ile Phe Arg 370 375 380 Gln AlaPhe 385 11 387 PRT Citrobacter freundii 11 Met Ser Tyr Arg Met Phe AspTyr Leu Val Pro Asn Val Asn Phe Phe 1 5 10 15 Gly Pro Asn Ala Ile SerVal Val Gly Glu Arg Cys Lys Leu Leu Gly 20 25 30 Gly Lys Lys Ala Leu LeuVal Thr Asp Lys Gly Leu Arg Ala Ile Lys 35 40 45 Asp Gly Ala Val Asp LysThr Leu Thr His Leu Arg Glu Ala Gly Ile 50 55 60 Asp Val Val Val Phe AspGly Val Glu Pro Asn Pro Lys Asp Thr Asn 65 70 75 80 Val Arg Asp Gly LeuGlu Val Phe Arg Lys Glu His Cys Asp Ile Ile 85 90 95 Val Thr Val Gly GlyGly Ser Pro His Asp Cys Gly Lys Gly Ile Gly 100 105 110 Ile Ala Ala ThrHis Glu Gly Asp Leu Tyr Ser Tyr Ala Gly Ile Glu 115 120 125 Thr Leu ThrAsn Pro Leu Pro Pro Ile Val Ala Val Asn Thr Thr Ala 130 135 140 Gly ThrAla Ser Glu Val Thr Arg His Cys Val Leu Thr Asn Thr Lys 145 150 155 160Thr Lys Val Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Ser Val 165 170175 Ser Ile Asn Asp Pro Leu Leu Met Leu Gly Lys Pro Ala Pro Leu Thr 180185 190 Ala Ala Thr Gly Met Asp Ala Leu Thr His Ala Val Glu Ala Tyr Ile195 200 205 Ser Lys Asp Ala Asn Pro Val Thr Asp Ala Ala Ala Ile Gln AlaIle 210 215 220 Arg Leu Ile Ala Arg Asn Leu Arg Gln Ala Val Ala Leu GlySer Asn 225 230 235 240 Leu Lys Ala Arg Glu Asn Met Ala Tyr Ala Ser LeuLeu Ala Gly Met 245 250 255 Ala Phe Asn Asn Ala Asn Leu Gly Tyr Val HisAla Met Ala His Gln 260 265 270 Leu Gly Gly Leu Tyr Asp Met Pro His GlyVal Ala Asn Ala Val Leu 275 280 285 Leu Pro His Val Ala Arg Tyr Asn LeuIle Ala Asn Pro Glu Lys Phe 290 295 300 Ala Asp Ile Ala Glu Phe Met GlyGlu Asn Thr Asp Gly Leu Ser Thr 305 310 315 320 Met Asp Ala Ala Glu LeuAla Ile His Ala Ile Ala Arg Leu Ser Ala 325 330 335 Asp Ile Gly Ile ProGln His Leu Arg Asp Leu Gly Val Lys Glu Ala 340 345 350 Asp Phe Pro TyrMet Ala Glu Met Ala Leu Lys Asp Gly Asn Ala Phe 355 360 365 Ser Asn ProArg Lys Gly Asn Glu Lys Glu Ile Ala Glu Ile Phe Arg 370 375 380 Gln AlaPhe 385 12 385 PRT Clostridium pasteurianum 12 Met Arg Met Tyr Asp PheLeu Ala Pro Asn Val Asn Phe Met Gly Ala 1 5 10 15 Gly Ala Ile Lys LeuVal Gly Glu Arg Cys Lys Ile Leu Gly Gly Lys 20 25 30 Lys Ala Leu Ile ValThr Asp Lys Phe Leu Arg Asn Met Glu Asp Gly 35 40 45 Ala Val Ala Gln ThrVal Lys Tyr Ile Lys Glu Ala Gly Ile Asp Val 50 55 60 Ala Phe Tyr Asp AspVal Glu Pro Asn Pro Lys Asp Thr Asn Val Arg 65 70 75 80 Asp Gly Leu LysVal Tyr Arg Lys Glu Asn Cys Asp Leu Ile Val Thr 85 90 95 Val Gly Gly GlySer Ser His Asp Cys Gly Lys Gly Ile Gly Ile Ala 100 105 110 Ala Thr HisGlu Gly Asp Leu Tyr Asp Tyr Ala Gly Ile Glu Thr Leu 115 120 125 Thr AsnPro Leu Pro Pro Ile Val Ala Val Asn Thr Thr Ala Gly Thr 130 135 140 GlySer Glu Val Thr Arg His Cys Val Ile Thr Asn Thr Lys Thr Lys 145 150 155160 Ile Lys Phe Val Ile Val Ser Trp Arg Asn Leu Pro Leu Val Ser Ile 165170 175 Asn Asp Pro Ile Leu Met Ile Lys Lys Pro Ala Gly Leu Thr Ala Ala180 185 190 Thr Gly Met Asp Ala Leu Thr His Ala Ile Glu Ser Tyr Val SerLys 195 200 205 Asp Ala Asn Pro Val Thr Asp Ala Leu Ala Ile Gln Ala IleLys Leu 210 215 220 Ile Ala Asn Asn Leu Arg Gln Ala Val Ala Leu Gly GluAsn Leu Glu 225 230 235 240 Ala Arg Glu Asn Met Ala Tyr Ala Ser Leu LeuAla Gly Met Ala Phe 245 250 255 Asn Asn Ala Asn Leu Gly Tyr Val His AlaMet Ala His Gln Leu Gly 260 265 270 Gly Leu Tyr Asp Met Ala His Gly ValAla Asn Ala Met Leu Leu Pro 275 280 285 His Val Glu Arg Tyr Asn Leu IleSer Asn Pro Lys Lys Phe Ala Asp 290 295 300 Ile Ala Glu Phe Met Gly GluAsn Ile Glu Gly Leu Ser Val Met Glu 305 310 315 320 Ala Ala Glu Lys AlaIle Asp Ala Met Phe Arg Leu Ser Lys Asp Val 325 330 335 Gly Ile Pro AlaSer Leu Lys Glu Met Gly Val Asn Glu Gly Asp Phe 340 345 350 Glu Tyr MetAla Lys Met Ala Leu Lys Asp Gly Asn Ala Phe Ser Asn 355 360 365 Pro ArgLys Gly Asn Glu Lys Asp Ile Val Lys Ile Phe Arg Glu Ala 370 375 380 Phe385

1. A mutant 1,3-propanediol dehydrogenase having an increased Km for1,3-propanediol over the corresponding wild-type 1,3-propanedioldehydrogenase Km for 1,3-propanediol.
 2. The mutant 1,3-propanedioldehydrogenase of claim 1 wherein the increased Km is about three timesthe wild-type Km.
 3. The mutant 1,3-propanediol dehydrogenase of claim 1having a Km of about 80 mM for 1,3-propanediol.
 4. The mutant1,3-propanediol dehydrogenase of claim 1 which is obtainable fromE.blattae having ATCC accession number PTA-92.
 5. The mutant1,3-propanediol dehydrogenase of claim 1 comprising a mutationcorresponding to residue His105 to Leu in E.blatte as shown in FIG. 3.6. The mutant 1,3 propanediol dehydrogenase of claim 1 comprising theamino acid having the sequence as shown in SEQ ID NO:2.
 7. An isolatednucleic acid encoding mutant 1,3-propanediol dehydrogenase having thesequence as shown in SEQ ID NO:2.
 8. The isolated nucleic acid of claim7 having the sequence as shown in SEQ ID No:1:
 9. An expression vectorcomprising the isolated nucleic acid of claim
 7. 10. A host cellcomprising the expression vector of claim
 9. 11. The host cell of claim10 that includes Citrobacter, Enterobacter, Clostridium, Klebsiella,Aerobacter, Lactobacillus, Aspergillus, Saccharomyces,Schizosaccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida,Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia,Salmonella, Bacillus, Streptomyces and Pseudomonas.
 12. The host cell ofclaim 10 wherein said mutant 1,3-propanediol dehydrogenase comprises amutation corresponding to residue His105 to Leu in E.blatte shown inFIG.
 3. 13. The host cell of claim 12 wherein said mutant1,3-propanediol dehydrogenase has the amino acid sequence as shown inSEQ ID NO:2.
 14. A method for making 1,3-propanediol in a microorganismcomprising the steps of i. obtaining a microorganism comprising a mutant1,3-propanediol dehydrogenase (PDD) having an increased Km for1,3-propanediol over the corresponding wild-type PDD, said microorganismcomprising at least one gene capable of expressing a dehydrataseactivity, and ii. contacting said microorganism with a carbon substrate.15. The method of claim 14 wherein said mutant 1,3-propanedioldehydrogenase comprises a mutation corresponding to residue His105 toLeu in E.blatte shown in FIG.
 3. 16. The method of claim 15 wherein saidmutant 1,3-propanediol comprises the amino acid sequence as shown in SEQID NO:2.
 17. The method of claim 14 wherein said mutant 1,3-propanedioldehydrogenase is obtainable from E.blattae having ATCC accession numberPTA-92.
 18. The method of claim 14 wherein said microorganism includesCitrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter,Lactobacillus, Aspergillus, Saccharomyces, Schizosaccharomyces,Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula,Debaryomyces, Mucor, Tolulopsis, Methylobacter, Escherichia, Salmonella,Bacillus, Streptomyces and Pseudomonas.
 19. An isolated microorganismhaving ATCC accession number PTA-92.